Bacillus thuringiensis (Bt) Cry proteins (also called δ-endotoxins or Cry toxins) are proteins that form a crystalline matrix in Bacillus that are known to possess insecticidal activity when ingested by certain insects. Over 180 holotype Cry proteins in 58 families have been identified and named. The various Cry proteins have been classified based upon their spectrum of activity and sequence homology. Prior to 1990, the major classes were defined by their spectrum of activity (Hofte and Whitely, 1989, Microbiol. Rev. 53:242-255), but more recently a new nomenclature was developed which systematically classifies the Cry proteins based on amino acid sequence homology rather than insect target specificities (Crickmore et al. 1998, Microbiol. Molec. Biol. Rev. 62:807-813).
Most Cry proteins active against lepidopteran insects are formed in the crystalline matrix as 130-140 kDa protoxins. In lepidopteran insects, the alkaline pH of the gut solubilizes the crystal and then gut proteases process the protoxin to toxic proteins of approximately 60-70 kDa. Processing of the protoxin to toxin has been reported to proceed by removal of both N- and C-terminal amino acids with the exact location of processing being dependent on the specific Cry protein and the specific insect gut fluids involved (Ogiwara et al., 1992, J. Invert. Pathol. 60:121-126). The proteolytic activation of a Cry protoxin can play a significant role in determining its specificity.
The three dimensional structure for several Cry proteins has been elucidated. The Cry3A protein, which is active against coleopteran insects, has three structural domains: the N-terminal domain I, from residues 58-290, consists of 7 alpha-helices, domain II, from residues 291-500, contains three beta-sheets in a so-called Greek key-conformation, and the C-terminal domain III, from residues 501-644, is a beta-sandwich in a so-called jellyroll conformation. The three dimensional structure for the lepidopteran active Cry1Aa toxin has also been solved (Grochulski et al., 1995, J. Mol. Biol. 254:447-464). The Cry1Aa toxin also has three domains: the N-terminal domain I, from residues 33-253, domain II from residues 265-461, and domain III from residues 463-609 with an additional outer strand in one of the β-sheets formed by residues 254-264. If the Cry3A and Cry1Aa structures are projected on other Cry1 sequences, domain I runs from about amino acid residue 28 to 260, domain II from about 260 to 460 and domain III from about 460 to 600. See, Nakamura et al., Agric. Biol. Chem. 54(3): 715-724 (1990); Li et al., Nature 353: 815-821 (1991); Ge et al., J. Biol. Chem. 266(27): 17954-17958 (1991); and Honee et al., Mol. Microbiol. 5(11): 2799-2806 (1991); each of which are incorporated herein by reference. Thus, it is now known that based on amino acid sequence homology, all Bt Cry proteins have a similar three-dimensional structure comprising three domains.
Based on the structure, a hypothesis has been formulated regarding the structure/function relationship of the Cry proteins. It is generally thought that domain I, the most N-terminal domain, is primarily responsible for pore formation in the insect gut membrane (Gazit & Shai, 1993, Appl. Environ. Microbiol. 57:2816-2820), domain II is primarily responsible for interaction with a gut receptor thereby determining toxin specificity (Ge et al., 1991, J. Biol. Chem. 32:3429-3436) and domain III, the most C-terminal domain, is most likely involved with protein stability (Li et al. 1991, supra) as well as having a regulatory impact on ion channel activity (Chen et al., 1993, PNAS 90:9041-9045). Domain III has also been implicated in determining specificity (U.S. Pat. No. 6,204,246, herein incorporated by reference). Swapping domain III between lepidopteran-active toxins, such as by in vivo recombination between the coding regions, can result in changes in specific activity. Binding experiments using such hybrids have shown that domain III is involved in binding to putative receptors of target insects, suggesting that domain III may have some impact on specificity through a role in receptor recognition.
The toxin portions of Bt Cry proteins are also characterized by having five conserved blocks across their amino acid sequence (Hofte & Whiteley, supra). Conserved block 1 (CB1) comprises approximately 29 amino acids. Conserved block 2 (CB2) comprises approximately 67 amino acids. Conserved block 3 (CB3) comprises approximately 48 amino acids. Conserved block 4 (CB4) comprises approximately 10 amino acids. Conserved block 5 (CB5) comprises approximately 12 amino acids. The sequences before and after these five conserved blocks are highly variable and thus are designated the “variable regions,” V1-V6. Domain I of a Bt Cry protein typically comprises a C-terminal portion of variable region 1, a complete conserved block 1, an entire variable region 2, and the N-terminal 52 amino acids of conserved block 2. Domain II typically comprises approximately the C-terminal 15 amino acids of conserved block 2, a variable region 3, and approximately the N-terminal 10 amino acids of conserved block 3. Domain III typically comprises approximately the C-terminal 38 amino acids of conserved block 3, variable region 4, conserved block 4, variable region 5, and conserved block 5. The Cry1 lepidopteran active toxins, among other Cry proteins, have a variable region 6 with approximately 1-3 amino acids lying within domain III.
Several Cry proteins, for example Cry1Ab, Cry1Ac, Cry1F and Cry2Ba have been expressed in transgenic crop plants and exploited commercially to control certain lepidopteran insect pests. For example, transgenic corn hybrids expressing a Cry1Ab protein have been available commercially for over 10 years. The Cry1Ab protein in these corn hybrids targets primarily European corn borer (Ostrinia nubilalis), the major lepidopteran pest in the US Corn Belt.
One concern raised regarding the deployment of transgenic crops expressing a Cry protein is whether insect pests will become resistant to the Cry protein. Insects have proven capable of developing resistance to Cry protein-containing products. Resistance in diamondback moth (Plutella xylostella) and other vegetable pests to commercial Bt microbial sprays, used extensively in organic farming, has developed in several parts of the world. One recent incidence of field resistance in a fall armyworm (Spodoptera frugiperda) population exposed to transgenic corn expressing Cry1F protein has been documented on the island of Puerto Rico (Storer et al. 2010. J. Econ. Entomol. 103:1031-1038). However, there have been no cases of any field failures in the United States associated with resistant field populations of corn or cotton pests exposed to transgenic crops since 1996 when transgenic crops expressing Cry proteins were first introduced.
The seed industry, university researchers and the US Environmental Protection Agency have worked together to develop management plans to help mitigate the onset of insect resistance. They are based primarily on a high dose and refuge strategy. A high dose strategy for European corn borer in corn, for example, is to use corn hybrids that express high enough levels of a Cry protein to kill even partially resistant European corn borers. The underlying hypothesis is that killing partially resistant ECB and preventing their mating greatly delays the development of resistance. The success of a high dose strategy depends in part on the specific activity of the Cry toxin to European corn borer and how much of that Cry toxin can be expressed in the transgenic corn plant. For example, the higher the specific activity of a Cry toxin to a pest, the less amount of Cry toxin is required to be expressed in a transgenic plant to achieve a high dose strategy. Because Cry1Ab is very toxic to European corn borer larvae (i.e. high specific activity) levels of expression of Cry1Ab that are achievable in transgenic plants easily places such corn hybrids in a high dose category.
Other possible ways to mitigate resistance development include pyramiding multiple Cry proteins in the same transgenic crop plant or replacing existing mature products with new products that produce different Cry proteins. For example, as the current Cry1Ab corn hybrid market matures, new products may be introduced that have Cry proteins other than Cry1Ab or other Cry proteins in addition to Cry1Ab. It would be beneficial for proteins that replace Cry1Ab to have the same or similar specific activity to European corn borer as Cry1Ab.
One candidate Cry toxin to replace Cry1Ab may be a Cry1Ba toxin. The holotype Cry1Ba toxin was first described by Brizzard et al. in 1988 (Nuc. Acids Res. 16:2723-2724). Subsequently, five other Cry1Ba toxins have been identified with each having about 99% identity to the holotype toxin. Cry1Ba toxins have been reported to have activity against certain lepidopteran pests, such as cabbage butterfly (Pieris brassicae), diamondback moth (Plutella xylostella), Egyptian cotton leafworm (Spodoptera littoralis), beet armyworm (Spodoptera exigua) and European corn borer (Ostrinia nubilalis). However, Cry1Ba has been reported to be greater than 2-fold less active against European corn borer than Cry1Ab (See for example, U.S. Pat. No. 5,628,995) and has been reported to have no activity against other major corn pests, for example corn earworm (Helicoverpa zea) (See for example, Karim et al. 2000. Pestic. Biochem. Physiol. 67: 198-216) and NAFTA populations of fall armyworm (Spodoptera frugiperda) (See for example, Monnerat et al. 2006. Appl. Environ. Microbiol. 72:7029-7035). One reason that Cry1Ba is not as active as Cry1Ab against at least European corn borer may be due to its lower solubility properties. Thus, there is a need to improve the specific activity of Cry1Ba against at least European corn borer and possibly expand its spectrum of activity to increase its potential as a replacement for Cry1Ab in transgenic corn.
The spectrum of insecticidal activity of an individual Cry toxin from Bt may be quite narrow, with a given Cry toxin being active against only a few species within an Order. For instance, the Cry3A protein is known to be very toxic to the Colorado potato beetle, Leptinotarsa decemlineata, but has very little or no toxicity to related beetles in the genus Diabrotica (Johnson et al., 1993, J. Econ. Entomol. 86:330-333). In addition, small variations in amino acid sequence within a Cry protein class can impact insecticidal activity. For example, von Tersch et al. (1991, Appl. Environ. Microbiol. 57:349-358) demonstrated that Cry1Ac proteins varying by seven amino acids showed significant differences in their spectrum of insecticidal activity. Although considered primarily lepidopteran-active toxins, Cry1Ba toxins have also been reported to be active against certain coleopteran insects pests including Colorado potato beetle (Leptinotarsa decemlineata), cottonwood leaf beetle (Chrysomela scripta) and coffee berry borer (Hypothenemus hampei).
Specificity of the Cry proteins is the result of the efficiency of the various steps involved in producing an active toxin protein and its subsequent interaction with the epithelial cells in the insect mid-gut. To be insecticidal, most known Cry proteins must first be ingested by the insect and proteolytically activated to form an active toxin. Activation of the insecticidal crystal proteins is a multi-step process. After ingestion, the crystals must first be solubilized in the insect gut. Once solubilized, the Cry proteins are activated by specific proteolytic cleavages. The proteases in the insect gut can play a role in specificity by determining where the Cry protein is processed. Once the Cry protein has been solubilized and processed it binds to specific receptors on the surface of the insects' mid-gut epithelium and subsequently integrates into the lipid bilayer of the brush border membrane. Ion channels then form disrupting the normal function of the midgut eventually leading to the death of the insect. There are stark differences in the solubility properties of the toxin portions of Cry proteins.
Certain lepidopteran-active Cry proteins have been engineered in attempts to improve specific activity or to broaden the spectrum of insecticidal activity. For example, the silk moth (Bombyx mori) specificity domain from Cry1Aa was moved to Cry1Ac, thus imparting a new insecticidal activity to the resulting chimeric protein (Ge et al. 1989, PNAS 86: 4037-4041). Also, Bosch et al. 1998 (U.S. Pat. No. 5,736,131), created a new lepidopteran-active toxin by substituting domain III of Cry1E with domain III of Cry1C thus producing a Cry1E-Cry1C hybrid toxin with a broader spectrum of lepidopteran activity.
There remains a need to design new and effective pest control agents that provide an economic benefit to farmers and that are environmentally acceptable. Needed are proteins with substantially altered properties, such as the engineered Cry1Ba proteins of the invention, that have greater specific activity than native Cry1Ba proteins against at least European corn borer, a major pest of corn in the United States, that may become resistant to existing insect control agents. Furthermore, engineered Cry1Ba proteins whose use minimizes the burden on the environment, as through transgenic plants, are desirable.
By increasing the specific activity of Cry1Ba to at least European corn borer, less Cry1Ba protein should be needed to be expressed in a maize plant therefore reducing the possible negative impacts of Cry1Ba on the plant. In addition, the increased specific-activity allows for use of the engineered Cry1Ba in a high dose strategy for ECB.